Human respiratory syncytial virus (RSV) is the most important viral agent responsible for severe respiratory tract disease among infants and children, resulting in approximately 100,000 hospitalizations and 5,000 deaths yearly in the United States (Chanock, R. M., Kim, H. W., Brandt, C. D. and Parrott, R. H., Viral infections of humans: epidemiology and control, pp. 471-489 (Evans, A. S., ed., 1982) (Plenum Publishing Corp., New York); Glezen, W. P., et al., (1986) Am. J. Dis. Chil. 140:543-546; MacDonald, N. E., et al., (1982) New England J. Med. 307:397-400; Stott, E. J. and Taylor, G., (1985) Arch. Virol., 84:1-52). Infants six weeks to nine months of age are most likely to develop bronchiolitis or pneumonia, with infants between two and seven months showing the peak incidence (Holberg, C. J., et al., (1991) Am. J. Epidemiol. 133:1135-1151).
About thirty percent of hospitalized young children with acute respiratory disease have RSV infection. In older children and adults the disease is milder. RSV appears to also be a major cause of morbidity and mortality in the elderly, equivalent to influenza. (Fleming, D. M. and Cross, K. W. (1993) Lancet 342:1507-1510). Infections with RSV are usually associated with fever, cough, runny nose, and fatigue, and are diagnosed clinically as bronchitis, bronchiolitis, pneumonia, croup, or viral infection. In older children and adults, the virus is generally limited to replication in the upper respiratory tract. Infants may be more severely involved when the virus extends into the lungs. Lung damage caused by RSV can be permanent.
RSV is a member of the order Mononegalovirales (Pringle, C. R., (1991) Arch. Virol. 117:137-140), which contains the families Paramyxoviridae, Rhabdoviridae, and Filoviridae. RSV belongs to the genus Pneumovirus of the Paramyxoviridae and exhibits the following structural characteristics. The genome consists of a nonsegmented, negative-sense RNA. This RNA is tightly wrapped in the viral proteins N (nucleocapsid protein), P (phosphoprotein), and L (polymerase), to form what is referred to as the nucleocapsid. The nucleocapsid is surrounded by a layer of M (matrix) proteins. This layer of M proteins is itself surrounded by a lipid membrane, in which the viral proteins G (glycoprotein), F (fusion), and SH (small hydrophobic) are embedded (Walsh, E. E. and Hruska, J., (1983) J. Virol., 47:171-177; Peeples, M. and Levine, S., (1979) Virol., 95:137-145).
Viral replication, in general, proceeds as follows. The glycoproteins in the viral membrane direct attachment of the virus to a target cell and direct fusion of the viral and cellular membranes. This is followed by dissociation (or uncoating) of the M proteins from the nucleocapsid and release of the nucleocapsid into the cell cytoplasm. Expression of the infecting nucleocapsid results in production of new nucleocapsids, and of M, G, F, and SH proteins. By a process which is poorly understood, the new nucleocapsids and M proteins become associated in regions of the target cell membrane in which the viral G, F, and SH proteins are embedded. This region of the cell membrane then buds, or pinches off, taking with it the nucleocapsid surrounded by M protein, and forms a new virion.
The exact intermolecular interactions which occur during the assembly of paramyxovirus virions in the infected cell is not known. It is believed that the M protein will be shown to play a central role in directing assembly of new virions, directing both its own self-assembly at the membrane and also colocalization of nucleocapsid and glycoprotein components. While there is no direct evidence of this for RSV, evidence supporting this belief has been generated for closely related viruses. In another paramyxovirus, Newcastle disease virus, the M protein can associate with membranes (Faaberg, K. S., and Peebles, M. E., (1988) Virology 166:123-132). The M protein of another paramyxovirus, Sendai virus, can self-associate (Heggeness, M. H., et al., Proc. Natl. Acad. Sci. USA (1982) 79:6232-6236). The M protein of the Sendai virus has also bee found in association with viral F protein in infected cells (Sanderson, C. M., et al., (1994) J. Virol. 68:69-76). These observations suggest that M protein of Sendai virus forms a bridge between nucleocapsids and glycoproteins located in the host cell membrane during virion assembly. This proposed model might also be applicable to other paramyxoviruses such as RSV.
Although the gene encoding RSV M protein had been cloned over a decade ago (Stake, M. and Venkatesan, S., (1984) J. Virol. 50:92-99), very little is known about the intermolecular interactions between M proteins or about the interaction of M protein with other RSV proteins. While it is believed that the M proteins of paramyxoviruses self-associate and mediate the association of nucleocapsids with nascent envelopes, it has not been known if RSV matrix proteins would physically interact directly in self-associating or if other proteins, cofactors, or processes were necessary to achieve this interaction. In particular, it was not known if the viral M2 protein (previously known as the 22K protein) played a role in the presumed interaction of the M protein with itself.
In order to combat the severe respiratory tract infections and disease in infants and children caused by RSV, there is a need to identify the protein--protein interactions of the RSV M protein and to develop a method for identifying compounds that modulate the M protein interactions to identify possible drug candidates to treat such severe infections. Compounds that inhibit M--M protein interaction may be effective antiviral agents if they prevent virus assembly. Compounds that promote M--M protein interaction may be effective antiviral agents if they prevent disassociation of matrix proteins after infection (uncoating).
For the first time, applicants have successfully expressed the RSV M protein in E. coli and yeast cells. Applicants have also purified the maltose binding protein-M fusion proteins for examination of protein--protein interaction in vitro and defined a specific protein--protein interaction between RSV M or fragments of the M protein in vivo. They have further demonstrated that the M protein, when fused separately with a transcription factor having a DNA binding domain and with the activation domain of a transcription factor, can interact within the complex milieu of a yeast cell in the absence of other viral proteins or unknown proteins or cofactors or processes supplied by the normal human cell host which are not supplied by the yeast cell host. Applicants have developed novel screening methods, including a novel high throughput yeast-based screening method (yeast two hybrid system), to identify agents that block or promote these specific protein--protein interactions. Agents identified by these new screening methods can be exploited as potential novel anti-RSV drugs with new modes of action. Additionally, the results provided by these novel screening methods will expand the understanding of the process of virus assembly which can be used to develop effective antiviral agents.